In the Drosophila leg disc, wingless (wg) and decapentaplegic (dpp) are expressed in a ventral-anterior and dorsal-anterior stripe of cells, respectively. This pattern of expression is essential for proper limb development. While the Hedgehog (Hh) pathway regulates dpp and wg expression in the anterior-posterior (A/P) axis, mechanisms specifying their expression in the dorsal-ventral (D/V) axis are not well understood. We present evidence that slimb mutant clones in the disc deregulate wg and dpp expression in the D/V axis. This suggests for the first time that their expression in the D/V axis is actively regulated during imaginal disc development. Furthermore, slimb is unique in that it also deregulates wg and dpp in the A/P axis. The misexpression phenotypes of slimb clones indicate that the regulation of wg and dpp expression is coordinated in both axes, and that slimb plays an essential role in integrating A/P and D/V signals for proper patterning during development. Our genetic analysis further reveals that slimb intersects the A/P pathway upstream of smoothened (smo).

Cells in Drosophila imaginal discs proliferate and organize in larval and pupal stages to form adult structures with specific patterns. Several secreted factors are responsible for coordinating the precise patterning of imaginal tissues for proper limb development. Hh, expressed in posterior cells induces neighboring anterior compartment cells to express their own anterior determinants: the Drosophila TGFβ homolog decapentaplegic (dpp), and the Wnt family member wingless (wg) (Lee et al., 1992; Basler and Struhl, 1994; Capdevila et al., 1994; Tabata and Kornberg, 1994; Felsenfeld and Kennison, 1995). In the leg imaginal disc, expression of dpp in the dorsal-anterior stripe is required for specification of dorsal structures, while wg in the ventral-anterior stripe determines ventral structures (Ferguson and Anderson, 1992; Struhl and Basler, 1993; Wilder and Perrimon, 1995). The expression patterns of dpp and wg are defined in both the anterior-posterior (A/P) and dorsal-ventral (D/V) axes.

The restricted domains of dpp and wg expression are tightly regulated in the A/P axis by the Hh/Ptc and PKA signaling pathways (Phillips et al., 1990; Ingham et al., 1991; Basler and Struhl, 1994; Capdevila and Guerrero, 1994; Tabata and Kornberg, 1994; Felsenfeld and Kennison, 1995; Li et al., 1995). Inactivation of ptc and pka or ectopic expression of hh induces ectopic dpp expression in the dorsal-anterior and ectopic wg expression in the ventral-anterior of the leg disc (Phillips et al., 1990; Ingham et al., 1991; Basler and Struhl, 1994; Jiang and Struhl, 1995; Li et al., 1995; Pan and Rubin, 1995) (Capdevila et al., 1994; Lepage et al., 1995). However, mutations in components of the A/P signaling pathway do not alter wg and dpp expression patterns in the D/V axis. To date no evidence exists of a D/V signaling pathway. It is possible that the D/V axis defined during embryogenesis is retained in imaginal tissues. One mechanism that prevents misexpression of wg in the dorsal and dpp in the ventral is the antagonistic relationship between wg and dpp. Inactivation of Wg or Dpp signaling leads to ectopic expression of dpp or wg, respectively (Brook and Cohen, 1996; Jiang and Struhl, 1996; Penton and Hoffman, 1996; Theisen et al., 1996).

To identify recessive overproliferation mutations in genes which are lethal in homozygous mutant animals, we have performed genetic screens in mosaic flies containing homozygous mutant patches in otherwise wild-type backgrounds (Xu et al., 1995). Two classes of recessive overproliferation mutations have been identified. Mutations of the first group cause mutant cells to undergo extensive proliferation and form unpatterned, tumorous outgrowths in mosaic adults. Mutations of the second group induce both patterned and irregular outgrowths. Here we report a new gene of the second class, slimb, which affects developmental signals that regulate cell proliferation and pattern organization. We present evidence that slimb mutant cells induce outgrowths by misexpressing wg and dpp. slimb regulates wg and dpp in both the A/P and D/V axes, demonstrating for the first time that these signals are coordinated. Genetic epistasis experiments reveal that slimb intersects A/P signaling upstream of smo.

slimb fly strains

122 excision lines were generated from two P-alleles (slmb00295 and slmb05415) and about half of them reverted to wild type. More than 30 excision alleles behaved as a single complementation group. Strong slmb alleles, including the original P alleles and slmbe4-1 (Fig. 2A), caused embryonic lethality while weak alleles caused larval and pupal lethality.

Cloning of slimb and H-slimb

Genomic DNA surrounding the P-insertion sites was obtained by plasmid rescue and used to isolate a genomic cosmid and cDNAs from an imaginal disc library. Comparison of genomic and cDNA sequences showed that slmb00295and slmb05415 inserted 150 nucleotides upstream of and within the coding region, respectively. Southern blot analysis of genomic DNA generated from the excision lines revealed that slmbe4-1 carries an approx. 3 kb deletion removing the 5′ end of the slmb transcript. The two P-alleles behave similarly to slmbe4-1 and are used interchangeably, while other excision alleles have weaker phenotypes. The 3.5 kb cDNA was sequenced to predict a protein product and cloned into the pCaSpeR-hs vector for germline transformation. Three of the transformant lines were able to fully rescue the lethality of the amorphic slmb alleles after 1 hour of heat shock every 24 hours during larval and pupal development. The human slmb homolog was identified by using the Drosophila cDNA as a probe to screen a human fetal brain library.

Generation and analysis of clones

Clones in adult flies and imaginal discs were generated by FLP-mediated mitotic recombination as previously described (Xu and Rubin, 1993; Xu and Harrison, 1994). Eggs from the appropriate crosses were collected for 24 hours and cultured at 25°C. Clones were induced in early second instar larvae by heat-shock induction of Flipase (38°C for 1 hour). Larvae from the following genotypes were used for clonal analysis: yw hsFLP1; P[FRT]82B P[πM]87E Sb63bP[y+]96E/P[FRT]82B slmbe4-1 or 00295 in a H1-1dpp-lacZ/+ background; and in a wg-lacZ/+ background. To detect hh-lacZ expression in slmb clones, hh-lacZ-P30 was recombined onto the slmb mutant chromosome and clones were induced in the following larvae: yw hsFLP1; P[FRT]82B P[πM]87E hh-lacZ-P30 P[πM]97E /P[FRT]82B slmb00295hh-lacZ-P30. Staining procedures followed standard protocols (Xu and Harrison, 1994).

Double mutant clones were induced in flies homozygous for the slmb null allele, but carried the hs-slmb31 rescue construct on the FRT40A chromosome arm. To ensure the full rescue of slmb− flies, eggs were collected every 24 hours and heat-shocked daily at 38°C for 60 minutes until hatched. Larvae of the following genotypes were generated and cultured at 25°C: yw hsFLP1; hs-slmb 31 P[FRT]40A/wgCX4ck P[FRT]40A; slmb00295/slmb00295, yw hsFLP1; hs-slmb 31 P[FRT]40A/dpp12ck P[FRT]40A; slmb00295/slmb00295, yw hsFLP1; P[FRT]82B P[πM]87E Sb63bP[y+]96E X/P[FRT]82B slmb00295hhrJ413, and yw hsFLP1; hs-slmb 31 P[FRT]40A/smoD16ck P[FRT]40A; slmb00295/slmb00295. slmb− clones were induced using the hs-slmb 31 P[FRT]40A chromosome at a frequency of 60% of discs. To verify that the smoD16ck P[FRT]40A chromosome that we used did not cause a cell-lethal phenotype, we examined clonal production by this chromosome and found it to produce smo− clones at a frequency of more than 25% of discs. In analyzing smoD16, slmb00295 double mutant clones, 40 mosaic leg discs were stained and found to have no ectopic wg or dpp expressed.

In a mosaic screen to identify recessive overproliferation mutations, we identified a new mutation, shiva, which causes outgrowths and disrupts pattern formation (Fig. 1) (Xu et al., 1995). In addition to two original P-insertion alleles, a deletion null allele (shivae4-1) was generated by excision of the P-elements and used for phenotypic analysis (Fig. 2A). Molecular and genetic characterization of shiva reveals that these mutations disrupt a single transcriptional unit and they can be rescued when the cDNA is expressed under control of the heat shock-inducible promoter (Materials and Methods). The transcript encodes a Cdc4-related protein containing F-box and WD-40 motifs. During preparation of this manuscript, Jiang and Struhl independently reported the identification of this gene as slimb (Jiang and Struhl, 1998). Thus, we are now renaming our gene slimb. Using a Drosophila slimb cDNA, we also isolated a human homolog (H-slimb) (Fig. 2B). The fly and human proteins share 78% amino acid identity throughout, suggesting that slimb is functionally conserved.

slimb induces outgrowths in mosaic adults

Phenotypic analysis revealed that slmb clones induce tissue outgrowths and supernumerary limbs in mosaic adults (Fig. 1). To analyze the slmb mosaic phenotype, the yellow (y) and Stubble+ (Sb+) cuticular markers were used to label slmb cells (Xu and Rubin, 1993; Materials and Methods). In mosaic legs, outgrowths are composed of slmb+ cells (y+ and Sb) (Fig. 1A-C). In addition to irregular outgrowths, supernumerary legs derived from slmb+ cells are also observed in slmb mosaic animals (Fig. 1B,C). Outgrowths are also observed in the wing blade (Fig. 1D,F), where mutant clones for slmb frequently produced small outgrowths in the adult wing (Fig. 1D). Similar to the leg outgrowths, the wing outgrowths consist of slmb+ cells (y+ and Sb) (Fig. 1E). Moreover, these outgrowths project from both the ventral and dorsal surfaces of the blade and occur in both the anterior and posterior halves of the wing (Fig. 1D). Outgrowths were organized into wing blade-like structures with wing margin bristles normally seen at the corresponding wing margin (Fig. 1D,E). Rarely, supernumerary wings consisting of symmetric duplications of anterior-most structures develop at the wing hinge (Fig. 1F). Although y (slmb) cells are rarely observed in mosaic animals, examination of mosaic discs revealed overproliferation of wild-type cells surrounding slmb mutant clones (Fig. 1G,H). These observations lead us to conclude that the mutant cells did not survive to adult stage, and that the adult outgrowths they induced are vestiges of their presence.

slimb regulates wg and dpp expression in both the A/P and D/V axes of the leg disc

slmb-induced outgrowths are reminiscent of the phenotypes caused by misexpression of dpp and wg (Struhl and Basler, 1993; Basler and Struhl, 1994; Wilder and Perrimon, 1995). Thus, we examined dpp and wg expression in slmb mosaic leg discs using wg-lacZ and dpp-lacZ reporter genes (Blackman et al., 1991; Kassis et al., 1992). slmb clones ectopically express both wg and dpp in a cell-autonomous fashion (Fig. 3). In respect to the A/P regions, 58/72 A clones and 9/31 P clones ectopically expressed wg, and 43/81 A clones and 6/17 P clones ectopically expressed dpp. A composite view of wg expression in the 103 analyzed slmb clones are illustrated in five subregions (Fig. 3J). dpp expression of 98 slmb clones was analyzed and not found to fall into any distinct domains. slmb mutant clones deregulate wg and dpp in both D/V and A/P axes. Ectopic wg expression is observed in both ventral and dorsal regions (Fig. 3A-C,J). Similar results are also observed for dpp (Fig. 3D-I). In slmb mutant clones situated within or near the endogenous dpp expression zone, dpp was expressed in the mutant cells but down-regulated in adjacent wild-type cells (Fig. 3G-I). Previously it had been shown that Wg and Dpp signaling mutually antagonize each other’s expression, which prevents expression of the two molecules in the same cells (Brook and Cohen, 1996; Jiang and Struhl, 1996; Penton and Hoffman, 1996; Theisen et al., 1996). Ectopic expression of both wg and dpp in slmb clones in the dorsal-anterior of the leg disc indicates a disruption of this mutual antagonism.

Although lacZ reporter genes may not always reflect protein expression, these reporter genes have been previously shown to serve as faithful indicators for wg and dpp gene expression in the leg disc (Jiang and Struhl, 1995; Li et al., 1995; Brook and Cohen, 1996; Jiang and Struhl, 1996; Penton and Hoffman, 1996; Lecuit and Cohen, 1997). To test whether ectopic wg and dpp expression are responsible for the outgrowth phenotype in slmb mosaic animals, we generated flies carrying clones of cells mutant for both slmb and wg, or slmb and dpp. In comparison to slmb mutant clones, double mutant clones do not cause any significant outgrowths (Table 1). Therefore, Wg and Dpp are two primary effector molecules responsible for the induction of outgrowths in slmb mosaic animals. These results are consistent with previous observations that wg and dpp are both required for defining the proximodistal outgrowth center (Diaz-Benjumea et al., 1994; Campbell and Tomlinson, 1995; Lecuit and Cohen, 1997).

slimb coordinates D/V and A/P signals to specify wg and dpp expression patterns

The slmb phenotype differs from those of all previously known genes, in that it is the first gene found to deregulate both wg and dpp expression in the D/V axis. Disrupting components of the Hh signaling pathway deregulates wg and dpp only along the A/P axis. For example, ectopic activation of hh or removal of ptc and pka results in misexpression of dpp and wg in anterior cells that normally do not express these genes. However, wg misexpression is always restricted to the ventral cells, while dpp misexpression is only in dorsal cells (Basler and Struhl, 1994; Jiang and Struhl, 1995; Li et al., 1995; Pan and Rubin, 1995). Thus, the control of wg and dpp expression in the D/V axis is not disrupted. The mechanism restricting wg and dpp in the D/V axis is not known. It is possible that the ability of dorsal cells to express dpp and of ventral cells to express wg is an inherent property of the D/V identity established during embryogenesis. The mutant phenotype of slmb clones in discs provides the first evidence that wg and dpp expression in the D/V axis is actively regulated during imaginal disc development, and is not solely defined during embryonic development. Since the Hh pathway regulates wg and dpp expression in the A/P axis, our results suggest that a pathway different from Hh may operate in imaginal discs to restrict their expression in the D/V axis (Fig. 4). This pathway cannot be either the Wg or Dpp signaling pathway since inactivation of Wg or Dpp signaling affects either dpp or wg expression, but not both (Brook and Cohen, 1996; Jiang and Struhl, 1996; Penton and Hoffman, 1996; Theisen et al., 1996). The slmb phenotypes described here were not observed in the previous study which used weak slmb alleles and revealed only A/P defects (Jiang and Struhl, 1998). The phenotypic differences probably reflect the fact that we have used a null allele instead of hypermorphic alleles.

In addition to D/V defects, slmb mutant clones also deregulate wg and dpp expression in the A/P axis. slmb is the first identified gene that regulates both wg and dpp expression in the A/P as well as D/V axes. The fact that mutations in slmb affect patterning in both axes suggests that the A/P and D/V signals are coordinated to specify wg and dpp expression patterns, and that slmb plays an essential role in integrating these signals (Fig. 4).

slimb intersects A/P signaling upstream of smo

To further explore how slmb regulation and function correlates with A/P signaling, we carried out double mutant analysis with slmb mutants and with mutants of hh and smo. No reduction of outgrowths was observed in slmb, hh double mutant clones (Table 1). Furthermore, slmb mutant clones have no effect on hh expression (Fig. 3K,L). This indicates that slmb acts downstream or independent of Hh signaling. In contrast, slmb, smo double mutant clones almost completely suppress slmb induced outgrowths (Table 1). Consistent with the adult phenotype, discs carrying slmb, smo clones fail to ectopically express either dpp or wg (Fig. 3M,N). These data suggest that slmb intersects the A/P signal upstream of smo (Fig. 4). The previous study suggested that slmb acts downstream of smo (Jiang and Struhl, 1998). This difference may be explained by the use of different alleles for smo and slmb. Many smo mutations are hypermorphic alleles which produce variable phenotypes (Alcedo et al., 1996; Heuvel and Ingham, 1996). smoD16 used in our analysis is caused by a DNA rearrangement which disrupts the smo transcript and produces the most severe embryonic phenotype (Alcedo et al., 1996; Heuvel and Ingham, 1996). The slmb product contains WD-40 repeats believed to act as a scaffold for the binding of multiple proteins (Neer et al., 1994; Sondek et al., 1996; Feldman et al., 1997; Skowyra et al., 1997). It is possible that this structure may allow for proteins such as Smo and components of a D/V pathway to converge. The Slmb-related protein Cdc4 from Saccharomyces cerevisiae along with Cdc53, and Cdc34 are part of the ubiquitin proteolysis machinery (Yochem and Byers, 1987; Goebl et al., 1988; Bai et al., 1996; Willems et al., 1996). Our data that Slmb acts upstream of Smo, together with its sequence homology with Cdc4, suggests that Slmb could be involved in the regulation of Smo protein degradation.

We thank members of our lab for helpful suggestions and discussion, U. Heberlein, D.J. Pan and the Berkeley Drosophila Genome Center for strains, S. Artavanis-Tsakonas for discussions, and J. Tamkun and A. Cowman for libraries. N. A. T., S. Z. and W. Y. W. were supported by NIH and Yale University predoctoral fellowships. This work was supported by grants from the Lucille P. Markey Charitable Trust and the NIH Cancer Institute to T. X.

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